Biotechnology

Features

• Students will study two important aspects of biotechnology using the alpha-amylase gene and alpha-amylase protein: 1) the nature of proteins and their action, and 2) the principles and methods for cloning and expressing a gene. Therefore, the analysis of protein and the analysis of DNA are integrated within a single project.
• The computer modeling software, RASMOL, is used in several lab exercises to study the three-dimensional structure of the alpha-amylase protein. Exercises in Appendix I will introduce students to some bioinformatics techniques that use sequence databases as well as sequence analysis software.
• Many of the techniques that are used at the beginning of the project will be repeated throughout the project. These recurring techniques will not only reinforce students' learning but will also show students that techniques can be used in different contexts to give different information.
• This project gives students an opportunity to apply biotechnology to a real, modern-day project. Not only will students need to evaluate the project in terms of the molecular biology but also in terms of practical considerations such as production and cost.

Examine Enzyme Activity of alpha-Amylase

1 Starch Plate Assay

2 Quantitative Enzyme Assay

3 Factors Affecting Enzyme Function

Examine alpha-Amylase Proteins

4 Analysis of Protein Structure Using RasMol

5 Analysis of alpha-Amylase Proteins

5A SDS-PAGE

5B Western Blotting

Examine DNA Structure

6 Analysis of DNA Structure Using RasMol

7 Isolation of Chromosomal DNA from Bacillus licheniformis

Find the alpha-Amylase Gene

8 PCR Amplification and Labeling of Probe DNA

9 Southern Hybridization

9A Restriction Enzyme Cleavage of Chromosomal DNA

9B Denaturation and Transfer of DNA to a Membrane

9C Southern Hybridization and Detection

Clone the alpha-Amylase Gene

10 Cloning the alpha-Amylase Gene

10A Cleavage of Chromosomal DNA

10B Cleavage of Plasmid DNA

10C Ligation of Chromosomal and Plasmid DNA

10D Transformation

10E Identification of alpha-Amylase Clones

Analyze alpha-Amylase Clones

11 Verification and Mapping of alpha-Amylase Clones

11A Verification of alpha-Amylase Clones Using PCR

11B Isolation of Plasmid DNA from alpha-Amylase Clones

11C Restriction Cleavage and Mapping of alpha-Amylase Plasmid DNA

11D Preservation of Recombinant Strains

11E Southern Analysis of alpha-Amylase Plasmid DNA

12 Enzyme Activity of alpha-Amylase Clones

Appendix I Additional Information and Exercises

Appendix II Frequently Used Procedures

Appendix III Bibliography